In acute hepatitis C virus (HCV) infection, an early HCV-specific T cell response is associated with viral clearance and recovery and we have recently shown that HCV-specific cytotoxic T cells (CTLs), the main effector cells for viral clearance, persist at least two decades after recovery from hepatitis C (Takaki et al. Nature Medicine, 6:578, 2000). These cellular immune responses were stronger in long-term recovered than in chronically infected individuals. In the latter, HCV-specific T cell responses appeared to be too weak to eliminate the virus, but sufficient to sustain liver inflammation. Whether this weakness was due to a low precursor frequency or to an impairment of effector functions of HCV specific T cells is not known. Detailed analysis of the functional properties of HCV-specific CD8+ T cells in chronically infected patients has often been hampered by the low precursor frequency and the requirement for extensive in vitro expansion of specific T cells. Quantification of HCV-specific CD8+ T cells was initially based on limiting dilution analysis of CTLs and thus, depended on the frequency of HCV specific cells, their in vitro expansion potential and their ability to lyse appropriate targets (Rehermann et al., J Clin Invest, 1996, 98:1432-1440). The introduction of MHC class I-peptide tetramers that bind to the T cell receptor of antigen specific cells now allows enumeration of specific CD8+ T cells regardless of their effector function and provides additional information on their phenotype if combined with staining for cell surface markers and intracellular cytokines. In this study, we used four HLA-A2 tetramers, specific for two HCV core and two HCV NS3 epitopes, and investigated the effector function of HCV-specific CD8+ T cells in 20 chronically infected and 12 long-term (5-13 years) recovered patients. HCV-specific, tetramer+ T cells were more frequently found in PBMC of chronically infected than recovered patients, but displayed an impaired proliferative capacity when stimulated in vitro with the specific HCV peptide. In contrast, tetramer+ cells of recovered patients expanded rapidly in the presence of HCV peptides. Furthermore, day 7 T cell lines of chronic patients displayed a lower specific cytotoxicity per tetramer+ cell than those of recovered patients. IFN-g-production of HCV-specific T cells was observed in only 17% of chronically infected patients but in 60% of recovered patients. Furthermore, in most chronically infected patients, less than half of the tetramer+ cells secreted cytokines upon specific stimulation. These impaired effector functions were associated with a CD45RO-, CD45RA+, CD28-, CD27? phenotype and were more pronounced in patients with weak ex vivo HCV-specific CD4+ T cell responses. Addition of recall antigen specific CD4+ T helper cells in vitro restored the proliferative function of a subpopulation (22 %) of tetramer+ cells. Thus, HCV-specific CD8+ T cells were functionally impaired in chronically infected patients with inadequate T helper cell responses. Both factors might contribute to viral persistence.